However, cotinine might not be responsible for releasing pro-angiogenic factors to stimulate the proliferation and invasion of choroidal capillaries in CNV. There are over 4,000 chemicals in the cigarette smoke. upregulated by the cotinine treatment, whereas expressions of epithelial-to-mesenchymal transition markers were downregulated. In conclusion, our study, for the first time, demonstrated that cotinine, rather than nicotine, affects the properties of RPE cells and genes6C8, are critical determinants. Other factors include cigarette smoking, which is the most consistently replicated and modifiable environmental risk factor for AMD development. Our earlier studies have shown that cigarette smokers are 1.76-fold more prone to develop AMD than non-smokers7, 8. In addition, cigarette GW 542573X smoking also interacts with rs11200638 polymorphism, superimposing the risk to 15.71 folds. Cigarette smoke contains over 4,000 chemicals. Nicotine is a key component and the determinant for addiction to smoking9, 10. Moreover, it is also the major component in different cigarette replacements, including the recently popular electronic cigarette and nicotine patches. In human primary fetal RPE GW 542573X culture, exposure of 1 1?M nicotine for 3 days changes RPE morphology without affecting cell proliferation, and reduces interleukin-8 (IL-8), metalloproteinase-2 (MMP2) and MMP9, but not vascular endothelial growth factor (VEGF) expression11. Similarly, exposure of 10?nM nicotine for 3 days did not induce ARPE-19 cell death or proliferation, but upregulated VEGF and downregulated PEDF expression12. In addition, one-day treatment of 10 or 0.1?mM nicotine did not affect ARPE-19 cell viability or caspase-3/7 activity13. Furthermore, nicotine (1, 10 or 100?M) did not induce cell death in porcine RPE during the 7-day treatment, but decreased VEGF secretion and reduced the phagocytotic ability of the RPE14. Under physiological conditions, Rabbit Polyclonal to GRAK nicotine, with a half-life of 2?hours, is continuously metabolized by hepatic cytochrome P450 enzyme CYP2A6 into cotinine15, which is the major metabolite of nicotine. The half-life of cotinine is 19?hours16. Plasma levels of nicotine and cotinine in daily cigarette smokers range from 0.08C0.15?M and 1.02C1.73?M, respectively17, 18. Cotinine has higher concentration and stays longer in the human body than nicotine. It possesses proven biological activities. Low concentration of cotinine (313?g/ml or below) stimulated secretory epithelial cell viability, whereas high concentration of cotinine (1250?g/ml or above) significantly decreased the total number of cells, metabolic activity as well as the secretory component19. Coherently, 10?nM cotinine is a mitogen for human vascular smooth muscle cells, but becomes toxic at higher concentrations20. However, the effect of cotinine on human RPE cells has yet to be examined. Here we hypothesized that cotinine has a more potent influence than nicotine on human RPE cells. We characterized the effects of continuous exposure of cotinine, with reference to nicotine, on the human RPE cells (ARPE-19), in terms of cell proliferation, cell apoptosis, cellular integrity, wound healing, angiogenic factor expression and phagocytotic activity. In addition, the mechanisms of the nicotine and cotinine effects were also investigated. Results The expression of nicotinic acetylcholine receptor subunits in ARPE-19 cells Nicotine has been shown to bind with the nicotinic acetylcholine receptor21. To understand the capability of ARPE-19 cells to respond to nicotine and cotinine, we examined the expression of nicotinic acetylcholine receptor gene by polymerase chain reaction (PCR). ARPE-19 cells highly expressed the 5 (and and and and were found. The expression of was dose-dependently and addictively downregulated across the nicotine and cotinine treatment groups, whereas the expression of was downregulated in the 1?M nicotine, 2?M cotinine and 1 and 2?M nicotine-cotinine mixture groups. The expression of was downregulated in the 1?M GW 542573X nicotine, 2?M cotinine as well as 2?M nicotine-cotinine mixture groups. *value)value)value)value)and and.